Modulating gamma-c-cytokine activity

ABSTRACT

Embodiments relate to peptide antagonists of  -family cytokines, which is associated with important human diseases, such as leukemia, autoimmune diseases, collagen diseases, diabetes mellitus, skin diseases, degenerative neuronal diseases and graft-versus-host disease (GvHD). Thus, inhibitors of  -cytokine activity are valuable therapeutic and cosmetic agents as well as research tools. Traditional approaches to inhibiting yc-cytokine activity involve raising neutralizing antibodies against each individual  -cytokine family member/′ receptor subunit. However, success has been limited and often multiple  -cytokine family members co-operate to cause the disease state. Combinatorial use of neutralizing antibodies raised against each factor is impractical and poses an increased risk of adverse immune reactions. The present embodiments overcome these shortcomings by utilizing peptide antagonists based on the consensus  -subunit binding site to inhibit  -cytokine activity. Such approach allows for flexibility in antagonist design. In several embodiments, peptides exhibit Simul-Block activity, inhibiting the activity of multiple  -cytokine family members.

PRIORITY AND CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/239,761, filed Oct. 9, 2015, which is hereby incorporated byreference in its entirety.

SEQUENCE LISTING IN ELECTRONIC FORMAT

The present application is being filed along with a Sequence Listing asan ASCII text file via EFS-Web. The Sequence Listing is provided as afile entitled BION006WOSEQLIST.txt, created and last saved on Oct. 6,2016, which is 5,981 bytes in size. The information in the electronicformat of the Sequence Listing is incorporated herein by reference inits entirety in accordance with 35 U.S.C. § 1.52(e).

FIELD

Some embodiments relate to peptide antagonists of γc-family cytokines, agroup of mammalian cytokines that are mainly produced by epithelial,stromal and immune cells and control the normal and pathologicalactivation of a diverse array of lymphocytes. Some embodiments alsorelate to the therapeutic uses of such peptides for the treatment ofcertain human diseases. The present embodiments also relate to thecosmeceutical applications of such peptides. Description of targetdiseases, cosmeceutical applications, as well as methods ofadministration, production, and commercialization of the peptides aredisclosed.

BACKGROUND

Cytokines are a diverse group of soluble factors that mediate variouscell functions, such as, growth, functional differentiation, andpromotion or prevention of programmed cell death (apoptotic cell death).Cytokines, unlike hormones, are not produced by specialized glandulartissues, but can be produced by a wide variety of cell types, such asepithelial, stromal or immune cells.

The γc-family cytokines are a group of mammalian cytokines that aremainly produced by epithelial, stromal and immune cells and control thenormal and pathological activation of a diverse array of lymphocytes.These cytokines are critically required for the early development of Tcells in the thymus as well as their homeostasis in the periphery.

SUMMARY

In some embodiments, a composite peptide is provided. In someembodiments, the composite peptide comprises amino acid sequences of atleast two interleukin (IL) protein gamma-c-box D-helix regions, whereinthe composite peptide comprises the amino acid sequenceP-K-E-F-L-E-R-F-V-H-L-V-Q-M-F-I-H-Q-S-L-S (SEQ ID NO: 3).

In some embodiments, the composite peptide of claim 1, wherein thecomposite peptide inhibits the activity of two or more γc-cytokinesselected from the group consisting of IL 2, IL 4, IL 7, IL 9, IL 15, andIL 21. In some embodiments, the composite peptide inhibits the activityof at least IL-15 and IL-21.

In some embodiments, the composite peptide further comprises a signalpeptide, in some embodiments, the composite peptide is furtherconjugated to one or more additional moieties at the N terminus, Cterminus or a side residue of the composite peptide. In some embodimentsof the composite peptide, the one or more additional moieties areselected from the group consisting of bovine serum albumin (BSA),albumin, Keyhold Limpet Hemocyanin (KLH), Fc region of IgG, a biologicalprotein that functions as scaffold, an antibody against a cell-specificantigen, a receptor, a ligand, a metal ion, and Poly Ethylene Glycol(PEG).

In some embodiments, the composite peptide consists of an amino acidsequence having at least 90% sequence identity with SEQ ID NO: 3. Insome embodiments, the composite peptide consists of an amino acidsequence having at least 95% sequence identity with SEQ ID NO: 3.

In some embodiments, a pharmaceutical composition is provided. In someembodiments, the pharmaceutical composition comprises a therapeuticallyeffective amount of a peptide conjugate, or a derivative thereof, and apharmaceutically acceptable carrier, diluent, excipient or combinationthereof, wherein the peptide conjugate or the derivative thereofmodulates the activity of two or more γc-cytokines selected from thegroup consisting of IL 2, IL 4, IL 7, IL 9, IL 15, and IL 21, whereinthe peptide conjugate comprises the amino acid sequenceP-K-E-F-L-E-R-F-V-H-L-V-Q-M-F-I-H-Q-S-L-S (SEQ ID NO: 3).

In some embodiments of the pharmaceutical composition, the peptideconjugate or the derivative thereof inhibits the activity of two or moreγc-cytokines selected from the group consisting of IL 2, IL 4, IL 7, IL9, IL 15, and IL 21.

In some embodiments of the pharmaceutical composition, the peptideconjugate or the derivative thereof further comprises an additionalconjugate at the N termini, C termini or a side residues thereof. Insome embodiments of the pharmaceutical composition, the peptideconjugate or the derivative thereof further comprises a signal peptide.

In some embodiments, the pharmaceutical composition further comprises aprotein that stabilizes the structure of the peptide conjugate or thederivative thereof and improves its biological activity, wherein theprotein is selected from the group consisting of bovine serum albumin(BSA), albumin, Fc region of immunoglobulin G (IgG), biological proteinsthat function as scaffold, Poly Ethylene Glycol (PEG), and derivativesthereof.

In some embodiments of the pharmaceutical composition, the derivativethereof comprises a peptide sequence sharing at least 90% identity withthe amino acid sequence of SEQ ID NO: 3. In some embodiments of thepharmaceutical composition, the derivative thereof comprises a peptidesequence sharing at least 95% identity with the amino acid sequence ofSEQ ID NO: 3.

In some embodiments, a method for blocking signaling by two or more γccytokine family members is provided. In some embodiments, the methodcomprises contacting a cell with a composite peptide and/or apharmaceutical composition provided herein.

In some embodiments, a method of inhibiting γc cytokine binding to a γcsubunit is provided. In some embodiments, the method comprisescontacting a γc subunit of a cell with a composite peptide and/or apharmaceutical composition provided herein.

In some embodiments, a method of treating a γc cytokine-mediated diseaseis provided. In some embodiments, the method comprises administering acomposite peptide and/or a pharmaceutical composition provided herein toa subject in need thereof, wherein the γc cytokine-mediated disease isselected from the group consisting of CD4 leukemia, CD8 leukemia, LGLleukemia, systemic lupus erythematosis, Sjögren's syndrome, Wegener'sgranulomatosis, Celiac disease, Hashimoto's thyroiditis, rheumatoidarthritis, diabetes mellitus, psoriasis, multiple sclerosis, uvietis,inflammation of the eye, and graft-versus-host disease (GvHD).

In some embodiments, a method of treating an HTLV-1-associatedmyelopathy (HAM)/tropical spastic paraparesis (TSP) associated diseaseis provided. In some embodiments, the method comprising administering acomposite peptide and/or a pharmaceutical composition provided herein toa subject in need thereof, wherein the HAM/TSP associated disease isselected from the group consisting of Adult T-cell Leukemia (ATL),HTLV-associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP), andother non-neoplastic inflammatory diseases associated with HTLV such asuveitis (HU), arthropathy, pneumopathy, dermatitis, exocrinopathy, andmyositis.

In some embodiments, a method of treating an inflammatory respiratorydisease is provided. In some embodiments, the method comprisingadministering a composite peptide and/or a pharmaceutical compositionprovided herein to a subject in need thereof, wherein the inflammatoryrespiratory disease is selected from the group consisting of asthma,sinusitis, hay fever, bronchitis, chronic obstructive pulmonary disease(COPD), allergic rhinitis, acute and chronic otitis, and lung fibrosis.

In some embodiments, a method of treating a cosmetic condition isprovided. In some embodiments, the method comprising administering acomposite peptide and/or a pharmaceutical composition provided herein toa subject in need thereof, wherein the cosmetic disease is selected fromthe group consisting of acne, hair loss, sunburn, nail maintenance, andappearance of aging.

In some embodiments, a pharmaceutical composition for use in treating acondition in a patient is provided. In some embodiments, thepharmaceutical composition comprises a therapeutically effective amountof a peptide conjugate, or a derivative thereof, and a pharmaceuticallyacceptable carrier, diluent, excipient or combination thereof, whereinthe peptide conjugate or the derivative thereof modulates the activityof two or more γc-cytokines selected from the group consisting of IL 2,IL 4, IL 7, IL 9, IL 15, and IL 21, wherein the peptide conjugatecomprises the amino acid sequenceP-K-E-F-L-E-R-F-V-H-L-V-Q-M-F-I-H-Q-S-L-S (SEQ ID NO: 3).

In some embodiments of the pharmaceutical composition, the derivativethereof comprises a peptide sequence sharing at least 90% identity withthe amino acid sequence of SEQ ID NO: 3. In some embodiments of thepharmaceutical composition, the derivative thereof comprises a peptidesequence sharing at least 95% identity with the amino acid sequence ofSEQ ID NO: 3.

In some embodiments, a pharmaceutical composition is provided for use intreating a γc cytokine-mediated disease, wherein the γccytokine-mediated disease is selected from the group consisting of CD4leukemia, CD8 leukemia, LGL leukemia, systemic lupus erythematosis,Sjögren's syndrome, Wegener's granulomatosis, Celiac disease,Hashimoto's thyroiditis, rheumatoid arthritis, diabetes mellitus,psoriasis, multiple sclerosis, uvietis, inflammation of the eye, andgraft-versus-host disease (GvHD).

In some embodiments, a pharmaceutical composition is provided for use intreating an HTLV-1-associated myelopathy (HAM)/tropical spasticparaparesis (TSP) associated disease, wherein the HAM/TSP associateddisease is selected from the group consisting of Adult T-cell Leukemia(ATL), HTLV-associated Myelopathy/Tropical Spastic Paraparesis(HAM/TSP), and other non-neoplastic inflammatory diseases associatedwith HTLV such as uveitis (HU), arthropathy, pneumopathy, dermatitis,exocrinopathy, and myositis.

In some embodiments, a pharmaceutical composition is provided for use intreating an inflammatory respiratory disease, wherein the inflammatoryrespiratory disease is selected from the group consisting of asthma,sinusitis, hay fever, bronchitis, chronic obstructive pulmonary disease(COPD), allergic rhinitis, acute and chronic otitis, and lung fibrosis.

In some embodiments, a pharmaceutical composition is provided for use intreating a cosmetic condition, wherein the cosmetic disease is selectedfrom the group consisting of acne, hair loss, sunburn, nail maintenance,and appearance of aging.

In some embodiments, a kit for treating a condition in a patient isprovided. In some embodiments of the kit, the condition is a γccytokine-mediated disease, an HTLV-1-associated myelopathy(HAM)/tropical spastic paraparesis (TSP) associated disease, aninflammatory respiratory disease, a cosmetic condition, or a combinationthereof, the kit comprising a pharmaceutical composition providedherein.

In some embodiments of the kit, the pharmaceutical composition comprisesa therapeutically effective amount of a peptide conjugate, or aderivative thereof, and a pharmaceutically acceptable carrier, diluent,excipient or combination thereof, wherein the peptide conjugate or thederivative thereof modulates the activity of two or more γc-cytokinesselected from the group consisting of IL 2, IL 4, IL 7, IL 9, IL 15, andIL 21, wherein the peptide conjugate comprises the amino acid sequenceP-K-E-F-L-E-R-F-V-H-L-V-Q-M-F-I-H-Q-S-L-S (SEQ ID NO: 3).

In some embodiments of the kit, the derivative thereof comprises apeptide sequence sharing at least 90% identity with the amino acidsequence of SEQ ID NO: 3. In some embodiments of the kit, the derivativethereof comprises a peptide sequence sharing at least 95% identity withthe amino acid sequence of SEQ ID NO: 3.

In some embodiments of the kit, the condition is one or more of CD4leukemia, CD8 leukemia, LGL leukemia, systemic lupus erythematosis,Sjögren's Sjoegren's syndrome, Wegener's granulomatosis, Celiac disease,Hashimoto's thyroiditis, rheumatoid arthritis, diabetes mellitus,psoriasis, multiple sclerosis, uvietis, inflammation of the eye,graft-versus-host disease (GvHD), Adult T-cell Leukemia (ATL),HTLV-associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP), andother non-neoplastic inflammatory diseases associated with HTLV such asuveitis (HU), arthropathy, pneumopathy, dermatitis, exocrinopathy,myositis, asthma, sinusitis, hay fever, bronchitis, chronic obstructivepulmonary disease (COPD), allergic rhinitis, acute and chronic otitis,and lung fibrosis, acne, hair loss, sunburn, nail maintenance, orappearance of aging.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows an alignment of the D-helix region of human γc-cytokinefamily members.

FIG. 1B depicts the γc-box (SEQ ID NO: 10) and IL-2/IL-15 box (SEQ IDNO: 11) motifs which give rise to the consensus sequence around theD-helix region of the γc-cytokines.

FIG. 2 depicts a diagramed representation of the biochemical propertiesof amino acids.

FIG. 3A shows inhibition of IL-15 and IL-9 activity by BNZ-γ in a PT-18proliferation assay.

FIG. 3B shows a proliferation assay of CTTL2 cells grown in the presenceof IL-2 or IL-15 and 0, 0.1, 1 or 10 uM BNZ-γ.

FIG. 3C shows inhibition of IL-15-mediated tyrosine-phosphorylation ofSTAT5 by BNZ-γ.

FIG. 4A shows an ex vivo T-cell proliferation assay using HAM/TSPperipheral blood. T-cell proliferation is inhibited by addition ofBNZ-γ.

FIG. 4B shows the population of CD4+CD25+ cells in an ex vivo T-cellproliferation assay using HAM/TSP peripheral blood is diminished afteradding BNZ-γ to the culture.

FIG. 4C shows the population of CD4+Ki67 cells in an ex vivo T-cellproliferation assay using HAM/TSP peripheral blood is reduced afteradding BNZ-γ to the culture.

FIG. 4D shows the percent of live cells by Guava staining in an ex vivoT-cell proliferation assay using HAM/TSP peripheral blood is notimpacted after adding BNZ-γ to the culture.

FIG. 5 shows the alignment of the sequence of SEQ ID NO: 3 to theD-helix regions of different human γc-cytokine family members. Theshaded areas represent amino acid sequences of the human γc-cytokinefamily members that are identical to their corresponding amino acids inthe sequence of SEQ ID NO: 3.

DETAILED DESCRIPTION Overview

More than 100 cytokines have been identified so far and are consideredto have developed by means of gene duplications from a pool ofprimordial genes (See Bazan. J. F. 1990, Immunol. Today 11:350-354). Insupport of this view, it is common for a group of cytokines to share acomponent in their multi-subunit receptor system. The mostwell-documented shared cytokine subunit in T cells is the common γsubunit (γc-subunit).

The γc-subunit is shared by 6 known cytokines (Interleukin-2 (IL-2),Interleukin-4 (IL-4), Interleukin-7 (IL-7), Interleukin-9 (IL-9),Interleukin-15 (IL-15), and Interleukin-21 (IL-21), collectively calledthe “γc-cytokines” or “γc-family cytokines”) and plays an indispensablerole in transducing cell activation signals for all these cytokines.Additionally, for each of the γc-cytokines, there are one or two privatecytokine-specific receptor subunits that when complexed with theγc-subunit, give rise to a fully functional receptor. (See Rochman etal., 2009, Nat Rev Immunol. 9: 480-90.)

The γc-family cytokines are a group of mammalian cytokines that aremainly produced by epithelial, stromal and immune cells and control thenormal and pathological activation of a diverse array of lymphocytes.These cytokines are critically required for the early development of Tcells in the thymus as well as their homeostasis in the periphery. Forexample, in the absence of the γc-subunit, T, B and NK cells do notdevelop in mice. (See Sugamura et al., 1996, Annu. Rev. Immunol.14:179-205).

The γc-cytokines are important players in the development of thelymphoid cells that constitute the immune system, particularly T, B, andNK cells. Further, γc-cytokines have been implicated in various humandiseases. Thus, factors that inhibit γc-cytokine activity would provideuseful tools to elucidate the developmental mechanism of subsets oflymphocytes and to treat immune disorders and γc-cytokine-mediateddiseases.

Germ line depletion of the genes encoding the γc-subunit in mice ormutations of γc-subunit in humans are known to cause severe combinedimmunodeficiency (SCID) by disrupting the normal appearance or functionof NK, T, and B cells. The importance of the γc-subunit in the signaltransduction of the γc-cytokines, IL-2, -4, -7, -9, 15, -21, isindicated in studies demonstrating the a of response of lymphocytes fromthese mice and human patients to the γc-cytokines (reviewed in Sugamuraet al., 1995 Adv. Immunol. 59:225-277). This indicates that disruptionof the interaction between the γc-subunit and a γc-cytokine wouldefficiently block the intracellular signaling events by the γc-cytokinefamily members. Therefore antagonist peptides according to the presentembodiments are expected to effectively block the pathogenic changes inhumans suffering from the diseases mediated by misregulation of theγc-cytokine family members.

As an alternative to antibody-mediated approaches for modulating theactivity of individual γc-cytokines, Applicants have devised novel, lowmolecular weight compounds herein referred to as “Simul-Block”, whichsuppress the activity of multiple γc-cytokines. These low molecularweight compounds, which include both chemicals and peptides, are lessimmunogenic than antibodies. These properties distinguish Simul-Block asa more efficient strategy for mediating γc-cytokine activity in clinicalinterventions.

Pathologies Associated with the γc-Cytokines

Recent studies have indicated that dysregulation of expression anddysfunction of the γc-cytokines could lead to a wide variety of humanimmunologic and hematopoietic diseases.

IL-2

While IL-2 was historically considered a prototype T cell growth factor,the generation of a knockout mouse lacking IL-2 expression revealed thatIL-2 is not critical for the growth or developmental of conventional Tcells in vivo. Over-expression of IL-2, however, leads to a preferentialexpansion of a subset of T-cells; the regulatory T cells (T-regs). (SeeAntony et al., 2006, J. Immunol. 176:5255-66.) T-regs suppress theimmune responses of other cells and thus act to maintain peripheraltolerance (reviewed in Sakaguchi et al., 2008, Cell 133:775-87).Breakdown of peripheral tolerance is thought to cause autoimmunediseases in humans.

Thus, the immunosuppressive function of T-regs is thought to prevent thedevelopment of autoimmune diseases (See Sakaguchi et al., 2008, Cell133:775-87). T-regs have also been implicated in cancer, where solidtumors and hematologic malignancies have been associated with elevatednumbers of T-regs (See Dc Rezende et al., 2010, Arch. Immunol. Ther.Exp. 58:179-190).

IL-4

IL-4 is a non-redundant cytokine involved in the differentiation of Thelper cells into the Th2 (T-helper type 2) subset, which promotes thedifferentiation of premature B cells into IgE producing plasma cells.IgE levels are elevated in allergic asthma. Thus, IL-4 is implicated inthe development of allergic Asthma. Antibodies targeting IL-4 can beused to treat or even prevent the onset of allergic asthma. (See LeBuanec et al., 2007, Vaccine 25:7206-16.)

IL-7

IL-7 is essential for B cell development and the early development of Tcells in the thymus. In mice, the abnormal expression of IL-7 causesT-cell-associated leukemia. (See Fisher et al., 1993, Leukemia2:S66-68.) However, in humans, misregulation of IL-7 does not appear tocause T-cell-associated leukemia. In humans, up-regulation of IL-7either alone or in combination with another γc-cytokine family member,IL-15, has been implicated in Large Granular Lymphocyte (LGL) leukemia.

IL-9

The role of IL-9 is still rather uncharacterized compared to otherγc-cytokine family members. Mice depleted of the IL-9 gene appear normaland do not lack any subsets of cells in the lymphoid and hematopoieticcompartments. Recent studies, however, reveal an in vivo role for IL-9in the generation of Th17 (T-helper induced by interleukin-17) cells(See Littman et al., 2010, Cell 140(6):845-58; and Nowak et al., 2009,J. Exp. Med. 206: 1653-60).

IL-15

IL-15 is critically involved in the development of NK cells, NK-T cells,some subsets of intraepithelial lymphocytes (IELs), γδ-T cells, andmemory-phenotype CD8 T-cells (See Waldmann, 2007, J. Clin. Immunol.27:1-18; and Tagaya et al., 1996, EMBO J. 15:4928-39.) Over-expressionof IL-15 in mice leads to the development of NK-T cell and CD8 cell typeT cell leukemia (See Fehniger et al., 2001, J. Exp. Med. 193:219-31:Sato et al. 2011 Blood in press). These experimentally induced leukemiasappear similar to LGL (large-granular lymphocyte) leukemia in humans,since in both instances the leukemic cells express CD8 antigen.

It is also suspected that IL-15-mediated autocrine mechanisms may beinvolved in the leukemic transformation of CD4 T lymphocytes. (See Azimiet al., 1998, Proc. Natl. Acad. Sci. 95:2452-7; Azimi et al., 1999, J.Immunol. 163:4064-72; Azimi et al., 2000, AIDS Res. Hum. Retroviruses16:1717-22; and Azimi et al., 2001, Proc. Natl. Acad. Sci. 98:14559-64).For example, CD4-tropic HTLV-I, which causes Adult T cell leukemia inhumans, induces autocrine growth of virus-transformed T cells throughthe production of IL-15 and IL-15Rα (Azimi et al., 1998, Proc. Natl.Acad. Sci. 95:2452-7).

In addition to leukemic transformation, recent studies implicate IL-15in the pathological development of Celiac disease (CD), an autoimmunedisease. IL-15 is known to stimulate the differentiation of NK, CD8 andintestinal intraepithelial lymphocyte (IEL) cells intolymphokine-activated killer (LAK) cells by inducing the expression ofcytolytic enzymes (i.e., Granzyme and Perforin) as well as interferon-γ.Celiac Disease (denoted CD from herein) is an immune-mediatedenteropathy that is triggered by the consumption of gluten-containingfood in individuals that express specific HLA-DQ alleles.

The prevalence of this disease is 1% in the western population. The onlycurrent treatment for CD is the complete elimination of gluten from thepatient's diet. The pathology of CD is mainly caused by extensive damageto the intestinal mucosa, which is caused by activated CD8 T cells thathave infiltrated to the intestinal lamina propria. These CD8 T cellsappear to be activated through mechanisms involving IL-15. One recentpublication demonstrated in mice that ectopic over-expression of IL-15by enterocytes leads to the development of enteropathy, which closelyresembles the lesions in CD patients. Neutralization of IL-15 activitydramatically diminished the pathological changes. Thus, an interventionblocking the activation of CD8 T cells by IL-15 appears to provide analternative strategy in managing CD to the conventional gluten-freediet.

IL-21

IL-21 is the most recently discovered member of the γc-family. Unlikeother family members, IL-21 does not appear to have potentgrowth-promoting effects. Instead, IL-21 is thought to function more asa differentiation factor than a factor controlling cellularproliferation (See Tagaya, 2010, J. Leuk. Biol. 87:13-15).

Current Strategies for Treating γc-Cytokine-Mediated Disorders

Because the γc-cytokines are thought to be involved in numerous humandiseases, several methods of treating γc-cytokine-implicated diseases byinhibiting γc-cytokine family activities have been proposed. Thesemethods include the use of cytokine-specific monoclonal antibodies toneutralize the targeted cytokine's activity in vivo; use of monoclonalantibodies targeting the private cytokine-specific receptor subunits(subunits other than the shared γc-subunit) to selectively inhibitcytokine activity: and use of chemical inhibitors that block thedownstream intracellular cytokine signal transduction pathway.

While cytokine-specific antibodies are often the first choice indesigning therapeutics, cytokines that share receptor components displayoverlapping functions (See Paul, W. E., 1989, Cell 57:521-24) and morethan one cytokine can co-operate to cause a disease (see exampledescribed below). Thus, approaches involving neutralization of a singlecytokine may not be effective in the treatment of cytokine-implicatedhuman diseases.

Strategies for designing therapeutics that inhibit the function ofmultiple cytokines via antibodies which recognize a shared receptorcomponent have also been proposed. However, the multi-subunit nature ofcytokine receptor systems and the fact that functional receptors for asingle cytokine can assume different configurations makes this approachdifficult.

For example, a functional IL-15 receptor can be either IL-15Rβ/γc orIL-15Rα/β/γc. (See Dubois et al., 2002, Immunity 17:537-47.) An antibodyagainst the IL-15Rβ receptor (TMβ1), is an efficient inhibitor of theIL-15 function, but only when the IL-15Rα molecule is absent from thereceptor complex. (See Tanaka et al., 1991, J. Immunol. 147:2222-28.)Thus, the effectiveness of a monoclonal anti-receptor antibody, whetherraised against a shared or a private subunit, can be context-dependentand is unpredictable in vivo.

Although clinical use of monoclonal antibodies against biologicallyactive factors or receptors associated with the pathogenesis of diseasesis an established practice, there are few demonstrations of successfuloutcomes. Moreover, establishment of a clinically-suited monoclonalantibody treatment is a long and difficult process, with the successfulgeneration of a neutralizing antibody largely a matter of luck. Forexample, due to the critical importance of the γc-subunit in mediatingsignaling by γc-family cytokines, many attempts to generate polyclonaland monoclonal antibodies against the γc-subunit have been made andthere exist many commercial antibodies recognizing the γc-subunit inmice and in humans. Curiously, however, none of these anti-γc-subunitantibodies block the function of the γc-cytokines.

Another problem with the therapeutic use of monoclonal antibodies isthat monoclonal antibodies are usually generated by immunizing rodentswith human proteins, so the generated antibody is a foreign protein andthus highly immunogenic. To circumvent this problem, the amino acidsequence of the monoclonal antibody is molecularly modified so that theantibody molecule is recognized as a human immunoglobulin (a processcalled humanization), but this process requires time and expense.

Targeting JAK3, as an Existing Alternative Example for the Inhibition ofMultiple γc-Cytokines

The interaction between the γc-subunit and a γc-cytokine leads to theactivation of an intracellular protein tyrosine kinase called Januskinase 3 (Jak3). Jak3, in turn, phosphorylates multiple signalingmolecules including STAT5, and PI3 kinase. The interaction of theγc-subunit and Jak3 is very specific. In fact, there is no otherreceptor molecule that recruits Jak3 for signal transduction. (SeeO'Shea, 2004, Ann. Rheum. Dis. 63:(suppl. 11):ii67-7.) Thus, theinhibition of cytokine signaling through the γc-subunit can beaccomplished by blocking the activity of Jak3 kinase. Accordingly,multiple chemical inhibitors that target the kinase activity of Jak3have been introduced to the market. (See Pesu et al., 2008, Immunol.Rev. 223:132-142.) One such example is CP690,550.

The major shortcoming of these protein kinase inhibitors is the lack ofspecificity to Jak3 kinase. These drugs intercept the binding of ATP(adenosine-triphosphate) molecules to Jak3 kinase, a common biochemicalreaction for many protein kinases, and thus tend to block the action ofmultiple intracellular protein kinases that are unrelated to Jak3 kinasewhose actions are critically needed for the well-being of normal cellsin various tissues. Thus, more specific inhibitors of signaling throughthe γc-subunit are needed.

There is therefore a great need for an alternative strategy for treatingγc-cytokine-implicated diseases.

Discovery of the γc-Box

The C-terminus (the D-helix) of the γc-cytokines contains the proposedsite for interacting with the common γc-subunit of the multi-unitcytokine receptors. (Bernard et al., 2004 J. Biol. Chem. 279:24313-21.)Comparison of the biochemical properties of the amino acids of allγc-cytokines identified in mice and humans revealed that the chemicalnature of the amino acids, for example, hydrophobicity, hydrophilicity,base/acidic nature, are conserved, if not identical, at many positionsin the D-helix across the members of the γc-cytokine family.

In contrast, the sequence of IL-13, which is related to the γc-cytokine,IL-4, but does not bind to the γc-subunit, does not exhibit significanthomology in the D-helix region to the γc-cytokines, suggesting that thesequence homology in the D-helix region is correlated with binding tothe γc-subunit. As shown in FIG. 1A, alignment of the amino acidsequences of the D-helix region of γc-cytokine family members in humansreveals a motif of moderate sequence homology in these cytokinesreferred to herein as “the γc-box”.

The γc-box (SEQ ID NO: 10) comprises 19 amino acids where out of the 19positions, positions 4, 5, and 13 are fully conserved as Phenylalanine,Leucine, and Glutamine, respectively. Less conservation is observed atpositions 6, 7 and 11 of the γc-box where the amino acid is one of twoor three related amino acids that share physico-chemical properties:position 6 may be occupied by the polar amino acids Glutamate.Asparagine or Glutamine; non-polar amino acids Serine or Arginine canoccupy position 7; and position 11 is occupied by either of thenon-polar aliphatic amino acids Leucine or Isoleucine. Positions 9 and16 may be occupied by the either the non-polar amino acid Isoleucine orthe polar amino acid Lysine. See FIG. 1B. Some differences in the aminoacid composition of the γc-box are observed at positions 9 and 16amongst subfamilies of the γc-cytokines. Comparison of the γc-cytokinesacross species indicates that Isoleucine is often present at the 9 and16 positions in the IL-2/15 subfamily, whereas the other γc-familymembers often possess Lysine in these positions. Not wishing to be boundby a particular theory, Isoleucine and Lysine are biochemicallydifferent and thus may impart specific conformational differencesbetween the IL-2/15 subfamily and other γc-cytokines.

Conservation of the γc-box motif between γc-cytokines is supported byfindings that an Glutamine (Gin, Q) residue located in the D-helixregion is critical for the binding of the γc-cytokines to theγc-subunit. (Bernard et al., 2004 J. Biol. Chem. 279: 24313-21.)

Peptide Inhibitors of γc-Cytokine Activity

The activity of γc-family cytokines may be blocked by disrupting theinteraction between the γc-cytokine and the γc-subunit, for example byintroducing a competitive inhibitor which can interact with theγc-subunit without stimulating signaling through the multi-subunitcytokine receptors. Not to be bound by a particular theory, theconserved γc-box motif, which participates in binding of the γc-familycytokines to the γc-subunit, presents a core base amino acid sequencewhich can be utilized to design peptide inhibitors of γc-cytokinesignaling.

The core γc-box amino acid sequence comprises:D/E-F-L-E/Q/N-S/R-X-I/K-X-L/I-X-Q (SEQ ID NO: 2) (where X denotes anyamino acid). At least some embodiments described herein relate to custompeptide derivatives of the core γc-box amino acid sequence which caninhibit the activity of one or more γc-cytokines. Custom peptidederivatives include any peptide whose partial amino acid sequence showsapproximately 50%, 50-60%, 60-70%, 70-80%, 80%, 90%, 95%, 97%, 98%, 99%or 99.8% identity to the core γc-box amino acid sequence. Custom peptidederivatives further include any peptide wherein a partial amino acidsequence of that peptide derivative comprises amino acids with similarphysico-chemical properties to the amino acids of the core γc-box. Forexample, amino acids with similar physico-chemical properties wouldinclude Phenylalanine, Tyrosine, Tryptophan, and Histidine, which arearomatic amino acids. FIG. 2 shows a diagrammed representation of aminoacids with similar physico-chemical properties which may be may besubstituted for the amino acids comprising the core γc-box. Peptidederivatives of the core γc-box may be 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 24, 25-30, 30-35, 35-40, 40-45, 45-50, or more than 50amino acids in length. In some embodiments, the custom peptidederivatives may be conjugated to the N-termini, C-termini and/or to theside residues of existing biological proteins/peptides.

Based on the identification of the conserved γc-box motif in cytokineswhich bind to the γc-subunit, Applicants have devised a novel, 19-mercustom derivative peptide which is an artificial composite peptidecombining the amino acid sequence of the human IL-2 and IL-15 γc-box.The 19-mer peptide, herein referred to as BNZ-γ, consists of the aminoacid sequence: I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO: 1),where the amino acids depicted by bold characters are conserved betweenIL-2 and IL-15 and the underlined amino acids represent positions wherethe physico-chemical properties of the amino acids are conserved.

Applicants discovered that the 19-mer BNZ-γ, suppresses IL-15 and IL-9induced cellular proliferation, but not IL-3 or IL-4 induced cellularproliferation. See FIG. 3A and EXAMPLE 2. Applicants furtherdemonstrated that BNZ-γ inhibits IL-15 mediated phosphorylation of theintracellular cytokine signal transduction molecule, STAT-5. See FIG. 3Cand EXAMPLE 5. These results demonstrate that custom peptide derivativesof the conserved γc-box motif can inhibit the activity of multipleγc-cytokines.

Several embodiments relate to custom derivative peptides of the 19-merBNZ-γ amino acid sequence, I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ IDNO: 1), which can inhibit the activity of one or more γc-cytokines.Custom peptide derivatives of the 19-mer BNZ-γ amino acid sequenceinclude any peptide whose partial amino acid sequence showsapproximately 50%, 50-60%, 60-70%, 70-80%, 80%, 90%, 95%, 97%, 98%, 99%or 99.8% identity to amino acid sequence:I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S(SEQ ID NO: 1). Custom peptidederivatives further include any peptide wherein a partial amino acidsequence of that peptide derivative comprises amino acids with similarphysico-chemical properties to the amino acids of sequence:I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S(SEQ ID NO: 1). In severalembodiments, the amino acid residues of the custom derivative peptidesretain similar physico-chemical properties with the amino acid residuesof BNZ-γ, but exhibit different biological inhibition specificity to the6 γc-cytokine family members from that of the original 19-mer peptide.Peptide derivatives of BNZ-γ may be 19, 20, 21, 22, 24, 25-30, 30-35,35-40, 40-45, 45-50, or more than 50 amino acids in length. In someembodiments, the custom peptide derivatives may be conjugated to theN-termini, C-termini and/or to the side residues of existing biologicalproteins/peptides.

Several embodiments relate to custom peptide derivatives of the γc-boxmotifs of IL-15, IL-2, IL-21, IL-4, IL-9, or IL-7, which are depicted inFIG. 1A. Other embodiments relate to custom derivative peptides whichare artificial composite peptides combining the amino acid sequence oftwo or more of the human IL-15, IL-2, IL-21, IL-4, IL-9, and IL-7 γc-boxmotifs. Several embodiments relate to custom peptide derivatives of theof the γc-box motifs of IL-15, IL-2, IL-21, IL-4, IL-9, or IL-7 having apartial amino acid sequence that shows approximately 50%, 50-60%,60-70%, 70-80%, 80%. 90%, 95%, 97%, 98%, 99% or 99.8% identity to aminoacid sequences of the of the γc-box motifs of IL-15, IL-2, IL-21, IL-4,IL-9, or IL-7. Custom peptide derivatives of the of the γc-box motifs ofIL-15, IL-2, IL-21, IL-4, IL-9, or IL-7 further include any peptidewherein a partial amino acid sequence of that peptide derivativecomprises amino acids with similar physico-chemical properties to theamino acids of sequence of the γc-box motifs of IL-15, IL-2, IL-21,IL-4, IL-9, or IL-7.

Several embodiments relate to custom peptide derivatives that wouldinhibit the function of one, all, or selective members of theγc-cytokines. In some embodiments, the custom peptide derivativesselectively target individual γc-cytokine family members. For example, acustom peptide derivative can selectively inhibit the function of IL-2,IL-4, IL-7, IL-9, IL-15, or IL-21. In other embodiments, a custompeptide derivative can inhibit 2 or more γc-cytokine family members.

For example, the custom peptide derivatives of the present embodimentscan selectively inhibit the function of IL-2 in combination with one ormore of IL-4, IL-7, IL-9, IL-15, and IL-21: IL-4 in combination with oneor more of IL-7, IL-9, IL-15, and IL-21; IL-7 in combination with one ormore of IL-9, IL-15, and IL-21; IL-9 in combination with one or more ofIL-2, IL-4, IL-7, IL-15, and IL-21; IL-15 in combination with one ormore of IL-2, IL-4, IL-7, IL-9, and IL-21: or IL-21 in combination withone or more of IL-2, IL-4, IL-7, IL-9, and IL-15. In other embodiments,custom peptide derivatives can comprehensively target all γc-cytokinefamily members.

Not wishing to be bound by a particular theory, the custom peptidederivatives can inhibit the function of all or selective members of theγc-cytokines by diminishing the binding of γc-cytokines to theγc-subunit, for example, as a competitive inhibitor. Such custom peptidederivatives may be used in diverse applications, including as a clinicaldrug.

Several embodiments relate to custom peptide derivatives that wouldmodulate (including enhance or reduce) the function of one, two, or moreof selective members of the γc-cytokines. In some embodiments, thecustom peptide derivatives selectively target individual γc-cytokinefamily members. For example, a custom peptide derivative can selectivelyenhance or inhibit the function of IL-2, IL-4, IL-7, IL-9, IL-15, orIL-21. In other embodiments, a custom peptide derivative can enhance orinhibit two or more γc-cytokine family members. In certain embodiments,custom peptide derivatives may compriseP-K-E-F-L-E-R-F-V-H-L-V-Q-M-F-I-H-Q-S-L-S (SEQ ID NO: 3), which canenhance or inhibit the activity of one, two or more of γc-cytokines. Incertain embodiments, custom peptide derivatives may compriseP-K-E-F-L-E-R-F-V-H-L-V-Q-M-F-I-H-Q-S-L-S (SEQ ID NO: 3), which caninhibit the activity of at least IL-15 and IL-21.

In some embodiments, custom peptide derivatives may include any peptidewhose partial amino acid sequence shows approximately 50%, 50-60%,60-70%, 70-80%, 80%, 90%, 95%, 97%, 98%, 99% or 99.8% identity to aminoacid sequence: P-K-E-F-L-E-R-F-V-H-L-V-Q-M-F-I-H-Q-S-L-S (SEQ ID NO: 3).Custom peptide derivatives further include any peptide wherein a partialamino acid sequence of that peptide derivative comprises amino acidswith similar physico-chemical properties to the amino acids of sequence:P-K-E-F-L-E-R-F-V-H-L-V-Q-M-F-I-H-Q-S-L-S (SEQ ID NO: 3).

In several embodiments, the amino acid residues of the custom derivativepeptides retain similar physico-chemical properties with the amino acidresidues of P-K-E-F-L-E-R-F-V-H-L-V-Q-M-F-I-H-Q-S-L-S (SEQ ID NO: 3),but exhibit different biological inhibition specificity to the 6γc-cytokine family members (i.e. IL-2, IL-4, IL-7, IL-9, IL-15, orIL-21) from that of the original peptide ofP-K-E-F-L-E-R-F-V-H-L-V-Q-M-F-I-H-Q-S-L-S (SEQ ID NO: 3). Peptidederivatives of the sequence of P-K-E-F-L-E-R-F-V-H-L-V-Q-M-F-I-H-Q-S-L-S(SEQ ID NO: 3) may be 19, 20, 21, 22, 24, 25-30, 30-35, 35-40, 40-45,45-50, or more than 50 amino acids in length.

In some embodiments, the custom peptide derivatives may be conjugated tothe N-termini, C-termini and/or to the side residues of existingbiological proteins/peptides. In some embodiments, the composite peptideof SEQ ID NO: 3 may be conjugated to other moieties through theN-terminus, C-terminus or side chains of the composite peptide. Theother moieties may include proteins or peptides that stabilize thecomposite peptide, or other moieties, including without limitation,bovine serum albumin (BSA), albumin, Keyhold Limpet Hemocyanin (KLH), Fcregion of IgG, a biological protein that functions as scaffold, anantibody against a cell-specific antigen, a receptor, a ligand, a metalion and Poly Ethylene Glycol (PEG).

The terms “oligopeptide,” “polypeptide,” “peptide,” and “protein” can beused interchangeably when referring to the custom peptide derivativesprovided in accordance with the present embodiments and can be used todesignate a series of amino acid residues of any length. Peptidesaccording to the present embodiments may also contain non-natural aminoacids. Linker elements can be joined to the peptides of the presentembodiments through peptide bonds or via chemical bonds. The peptides ofthe present embodiments may be linear or cyclic, and may include (D) aswell as (L) amino acids.

Peptides of the present embodiments may also contain one or more rareamino acids (such as 4-hydroxyproline or hydroxylysine), organic acidsor amides and/or derivatives of common amino acids, such as amino acidshaving the C-terminal carboxylate esterified (e.g., benzyl, methyl orethyl ester) or amidated and/or having modifications of the N-terminalamino group (e.g., acetylation or alkoxycarbonylamino), with or withoutany of a wide variety of side chain modifications and/or substitutions(e.g., methylation, benzylation, t-butylation, tosylation,alkoxycarbonylamino, and the like).

Residues other than common amino acids that may be present include, butare not limited to, penicillamine, tetramethylene cysteine,pentamethylene cysteine, mercaptopropionic acid,pentamethylene-mercaptopropionic acid, 2-mercaptobenzene,2-mercaptoaniline, 2-mercaptoproline, ornithine, diaminobutyric acid,aminoadipic acid, m-aminomethylbenzoic acid, and diaminopropionic acid.

Peptides of the present embodiments can be produced and obtained byvarious methods known to those skilled in the art. For example, thepeptide may be produced by genetic engineering, based on the nucleotidesequence coding for the peptide of the present embodiments, orchemically synthesized by means of peptide solid-phase synthesis and thelike, or produced and obtained in their combination.

One skilled in the art can synthesize the custom peptide derivativesbased on the present disclosure of the conserved γc-box motif andknowledge of the biochemical properties of amino acids as described inFIG. 2. Some embodiments also relate to polynucleotides comprisingnucleotide sequences encoding the peptides of the present invention.“Nucleotide sequence,” “polynucleotide,” or “nucleic acid” can be usedinterchangeably, and are understood to mean either double-stranded DNA,a single-stranded DNA or products of transcription of the said DNAs(e.g., RNA molecules). Polynucleotides can be administered to cells orsubjects and expressed by the cells or subjects, rather thanadministering the peptides themselves. Several embodiments also relateto genetic constructs comprising a polynucleotide sequence encoding thepeptides of the present invention. Genetic constructs can also containadditional regulatory elements such as promoters and enhancers and,optionally, selectable markers.

Methods of Treating γc-Cytokine Mediated Diseases

Several embodiments relate to the use of γc-antagonist peptides in thetreatment of γc-cytokine mediated diseases. Use of custom peptidederivative according to the present embodiments allows for flexibilityin the design of the therapeutic agent (custom design of the peptide)and enables more comprehensive outcomes, which would not be accomplishedby conventional strategies employing anti-cytokine or anti-cytokinereceptor antibodies.

Described herein is a novel method of blocking the action of γc-familycytokines. Such manipulations can yield effective methods of clinicalinterventions in treating diseases related to the dysregulation ordysfunction of γc-cytokines. Examples of disease that may be treated bydisrupting the interaction between the γc-cytokine and the γc-subunitinclude autoimmune diseases such as systemic lupus erythematosis,Sjögren's syndrome. Wegener's granulomatosis Celiac disease. Hashimoto'sor auto-immune thyroiditis; collagen diseases including rheumatoidarthritis, inflammatory bowel disease, diabetes mellitus, autoimmunediseases of the skin such as psoriasis; degenerative neuronal diseasessuch as multiple sclerosis, uvietis or inflammation of the eye andsympathetic ophthalmia, graft-versus-host disease (GvHD) and myastheniagravis.

In some embodiments, the γc-antagonist peptides described herein may beused in the treatment of 1-Human T-cell Lymphotropic type I and II(HTLV-I and HTLV-II)-associated diseases including Adult T-cell Leukemia(ATL), HTLV-associated Myelopathy/Tropical Spastic Paraparesis(HAM/TSP), and other non-neoplastic inflammatory diseases associatedwith HTLV such as uveitis (HU), arthropathy, pneumopathy, dermatitis,exocrinopathy and myositis. In some embodiments, the γc-antagonistpeptides described herein may be used in the treatment of other viraldiseases such as influenza. AIDS, HBV and Herpes or parasitic diseases.

In several embodiments, the γc-antagonist peptides may be administeredbefore, during, and or after transplantation of various organs as animmunosuppressant agent.

In some embodiments, the γc-antagonist peptides described herein may beused in the treatment of immune-mediated diseases such as asthma andother inflammatory respiratory diseases, such as, but not limited tosinusitis, hay fever, bronchitis, chronic obstructive pulmonary disease(COPD), allergic rhinitis, acute and chronic otitis, lung fibrosis. Insome embodiments. γc-antagonist peptides may be administered to treat orprevent allergic reactions due to exposure to allergens, chemical agentsor other common causes of acute respiratory disease. In someembodiments, γc-antagonist peptides may be administered to treat orprevent inflammatory responses caused by viruses, bacteria, chemicalreagents, and biochemical reagents.

In several embodiments, the γc-antagonist peptides may be administeredto treat some types of malignancies such as LGL-leukemia,Intraepithelial lymphoma and leukemia in Refractory Celiac Disease, NKleukemia/lymphoma and NK-T leukemia/lymphoma

In some embodiments, custom peptide derivatives according to theembodiments described herein can be used for cosmetic purposes, such asthe treatment of acne, hair loss, sunburn and nail maintenance, includedto ointment as anti-aging component because of the anti-inflammatorynature of them.

Several embodiments relate to therapeutic antagonist peptides that wouldinhibit the function of all or selective members of the γc-cytokines. Insome embodiments, therapeutic antagonist peptides selectively inhibitindividual γc-cytokine family members (custom peptides). In otherembodiments, therapeutic antagonist peptides can comprehensively inhibitall γc-cytokine family members (Simul-Block). In some embodiments,therapeutic antagonist peptides selectively inhibit subsets of theγc-cytokines. Not wishing to be bound by a particular theory, thepeptide antagonists can inhibit the function of all or selective membersof the γc-cytokines by diminishing the binding of γc-cytokines to theγc-subunit, for example, as a competitive inhibitor.

Several members of the γc-cytokine family, IL-2, IL-7, and IL-15, butnot IL-4 have been implicated as being involved in graft versus hostdisease (GvHD) in an experimental mouse model. (Miyagawa et al., 2008 J.Immunol. 181:1109-19.) One embodiment relates to the use of therapeuticantagonist peptides that selectively inhibit IL-2, IL-7, and IL-15activity for the treatment of GvHD in humans, allowing survival of thegrafted tissues or bone marrow cells. Other embodiments relate to theuse of therapeutic antagonist peptides that selectively inhibit acombination of IL-2 and IL-7, IL-2, and IL-15, or IL-7 and IL-15 totreat GvHD. Other embodiments relate to the use of a combination oftherapeutic antagonist peptides that selectively inhibit IL-2, IL-7, orIL-15.

Some embodiments relate to the use of therapeutic antagonist peptidesthat selectively inhibit IL-2 function for the treatment of autoimmunedisorders where T-regs have been implicated as playing a role. In someembodiments, peptide-mediated inhibition of T-regs can enhance thenatural anti-cancer immunity in humans, providing a novel means ofanti-cancer therapy.

Several embodiments relate to the use of therapeutic antagonist peptidesthat selectively inhibit IL-4 to treat asthma.

Some embodiments relate to the use of therapeutic antagonist peptidesthat selectively inhibit IL-7 either alone or in combination withtherapeutic antagonist peptides that selectively inhibit the γc-cytokinefamily member, IL-15, as a therapeutic agent for LGL leukemia. In someembodiments therapeutic antagonist peptides that selectively inhibitboth IL-7 and IL-15 activity can be used to treat LGL leukemia. Severalembodiments relate to the use of BNZ-γ to treat LGL leukemia. In someembodiments, specific γc-antagonist peptides that selectively IL-15alone or specific γc-antagonist peptides that selectively IL-15 and IL-7are used as a therapeutic agent for CD4/CD8 T lymphocyte-associatedleukemia including that caused by the HTLV-I.

Several embodiments relate to the use of γc-antagonist peptides thatselectively inhibit the activity of IL-9, either alone or in combinationwith the other γc-cytokine family members, as a therapeutic agent forhuman diseases that involve the abnormal development of Th17 cells.

Several embodiments relate to the use of therapeutic antagonist peptidesthat selectively inhibit IL-15 activity as a therapeutic agent fortreating CD. One recent publication suggested that IL-21, in addition toIL-15, may play a role in CD pathogenesis. (See Bodd et al., 2010,Mucosal Immunol. 3:594-601.) This suggests that optimum treatment of CDby conventional anti-cytokine or cytokine-receptor antibodies wouldbenefit from a combination of at least two antibodies recognizingcomponent that belong to the IL-15 and IL-21 systems. In someembodiments, custom derivative antagonist peptides that selectivelyinhibit both IL-15 and IL-21 activity are used as a therapeutic agentfor treating CD.

In addition to having therapeutic applications, γc-antagonist peptideshave applications in consumer products as well. Several embodimentsrelate to the use of γc-antagonist peptides in skin care products suchas anti-aging, anti-inflammatory, anti-acne, and other relatedapplications. Some embodiments relate to the use of γc-antagonistpeptides in hair products as anti-hair loss ingredient to treat hairloss caused by autoimmune disorders.

Another embodiment relates to the development of chemical compounds(non-peptide, non-protein) that have a spatial structure which resemblesthe 19-mer amino acid sequence I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S(SEQ ID NO: 1) and can fit into the pocket of the γc-subunit tostructurally hinder the access of a γc-cytokine to the γc-subunit forbinding. Some embodiments relate to the use of structurally similarchemical compounds as inhibitors of γc-cytokine activity. Such molecularmimicry strategy to further refine the development of syntheticcompounds resembling in structure to existing biologicalpeptide/proteins is described in Orzaez et al., 2009 Chem. Med. Chem.4:146-160. Another embodiment relates to administration of chemicalcompounds (non-peptide, non-protein) that have a resembling 3D structureas the 19-mer amino acids sequence I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S(SEQ ID NO: 1) to treat γc-cytokine-mediated diseases.

Several embodiments relates to the administration of a peptide of aminoacid sequence I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO: 1) totreat γc-cytokine-mediated diseases. Another embodiment relates to theadministration of derivative peptides of amino acid sequenceI-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S(SEQ ID NO: 1), wherein the aminoacid sequence of the derivative peptide has similar physico-chemicalproperties as a peptide of the amino acid sequenceI-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO: 1), but has distinctbiological activity, to treat γc-cytokine-mediated diseases. Anotherembodiment relates to administration of a peptide of amino acid sequenceI-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO: 1) conjugated to theN- and C-termini or to the side residues of existing biologicalproteins/peptides into patients to treat γc-cytokine-mediated diseases.

Several embodiments relate to administration of polyclonal andmonoclonal antibodies raised against a peptide comprising of amino acidsequence I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO: 1) intopatients as an immunogen to treat γc-cytokine-mediated diseases. Anotherembodiment relates to administration of polyclonal and monoclonalantibodies that were raised against derivative peptides of amino acidsequence I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO: 1) whereinthe amino acid sequence of the derivative peptide has similarphysico-chemical properties as a peptide of the amino acid sequence1-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO: 1), but has distinctbiological activity, into patients as an immunogen to treatγc-cytokine-mediated diseases.

Administration of γc-Antagonist Peptides

The present embodiments also encompass the use of γc-antagonist peptidesfor the manufacture of a medicament for the treatment of a disease. Thepresent embodiments also encompass a pharmaceutical composition thatincludes γc-antagonist peptides in combination with a pharmaceuticallyacceptable carrier. The pharmaceutical composition can include apharmaceutically acceptable carrier and a non-toxic therapeuticallyeffective amount of γc-antagonist peptides, or other compositions of thepresent embodiments.

The present embodiments provide methods of using pharmaceuticalcompositions comprising an effective amount of antagonists forγc-cytokines in a suitable diluent or carrier. A γc-antagonist of thepresent embodiments can be formulated according to known methods used toprepare pharmaceutically useful compositions. A γc-antagonist can becombined in admixture, either as the sole active material or with otherknown active materials, with pharmaceutically suitable diluents (e.g.,phosphate, acetate, Tris-HCl), preservatives (e.g., thimerosal, benzylalcohol, parabens), emulsifying compounds, solubilizers, adjuvants,and/or carriers such as bovine serum albumin.

Suitable carriers and their formulations are described in Remington'sPharmaceutical Sciences, 16^(th) ed. 1980 Mack Publishing CO.Additionally, such compositions can contain a γc-antagonist complexedwith polyethylene glycol (PEG), metal ions, or incorporated intopolymeric compounds such as polyacetic acid, polyglycolic acid,hydrogels etc., or incorporated into liposomes, microemulsions,micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, orspheroblasts. Such compositions will influence the physical state,solubility, stability, rate of in vivo release, and rate of in vivoclearance or a γc-antagonist. A γc-antagonist can be conjugated toantibodies against cell-specific antigens, receptors, ligands, orcoupled to ligands for tissue-specific receptors.

Methods of administrating γc-antagonists of the present embodiments maybe selected as appropriate, depending on factors, such as the type ofdiseases, the condition of subjects, and/or the site to be targeted. Theγc-antagonists can be administered topically, orally, parenterally,rectally, or by inhalation. The term “parenteral” includes subcutaneousinjections, intravenous, intramuscular, intraperitoneal, intracisternalinjection, or infusion techniques. These compositions will typicallyinclude an effective amount of a γc-antagonist, alone or in combinationwith an effective amount of any other active material.

The amount of the peptide contained in pharmaceutical compositions ofthe present embodiments, dosage form of the pharmaceutical compositions,frequency of administration, and the like may be selected asappropriate, depending on factors, such as the type of diseases, thecondition of subjects, and/or the site to be targeted. Such dosages anddesired drug concentrations contained in the compositions may varyaffected by many parameters, including the intended use, patient's bodyweight and age, and the route of administration. Pilot studies willfirst be conducted using animal studies and the scaling to humanadministration will be performed according to art-accepted practice.

In one embodiment, host cells that have been genetically modified with apolynucleotide encoding at least one γc-antagonist peptide areadministered to a subject to treat a proliferation disorder and/or toreduce the growth of malignant cells. The polynucleotide is expressed bythe host cells, thereby producing the peptides within the subject.Preferably, the host cells are allogeneic or autogenic to the subject.

In a further aspect, γc-antagonist peptides can be used in combinationwith other therapies, for example, therapies inhibiting cancer cellproliferation and growth. The phrase “combination therapy” embraces theadministration of γc-antagonist peptides and an additional therapeuticagent as part of a specific treatment regimen intended to provide abeneficial effect from the co-action of these therapeutic agents.Administration of these therapeutic agents in combination typically iscarried out over a defined time period (usually minutes, hours, days orweeks depending upon the combination selected).

A combination therapy is intended to embrace administration of thesetherapeutic agents in a sequential manner, that is, wherein eachtherapeutic agent is administered at a different time, as well asadministration of these therapeutic agents, or at least two of thetherapeutic agents, in a substantially simultaneous manner.Substantially simultaneous administration can be accomplished, forexample, by administering to the subject a single capsule having a fixedratio of each therapeutic agent or in multiple, single capsules for eachof the therapeutic agents. Sequential or substantially simultaneousadministration of each therapeutic agent can be effected by anappropriate route including, but not limited to, oral routes,intravenous routes, intramuscular routes, and direct absorption throughmucous membrane tissues. There therapeutic agents can be administered bythe same route or by different routes. The sequence in which thetherapeutic agents are administered is not narrowly critical.

Combination therapy also can embrace the administration of thetherapeutic agents as described above in further combination with otherbiologically active ingredients (such as, but not limited to, a secondand different therapeutic agent) and non-drug therapies (such as, butnot limited to, surgery or radiation treatment). Where the combinationtherapy further comprises radiation treatment, the radiation treatmentmay be conducted at any suitable time so long as a beneficial effectfrom the co-action of the combination of the therapeutic agents andradiation treatment is achieved. For example, in appropriate cases, thebeneficial effect is still achieved when the radiation treatment istemporarily removed from the administration of the therapeutic agents,perhaps by days or even weeks.

In certain embodiments, γc-antagonist peptides can be administered incombination with at least one anti-proliferative agent selected from thegroup consisting of chemotherapeutic agent, an antimetabolite, andantitumorgenic agent, and antimitotic agent, and antiviral agent, andantineoplastic agent, an immunotherapeutic agent, and a radiotherapeuticagent.

In certain embodiments, γc-antagonist peptides can be administered incombination with at least one anti-inflammatory agent selected from thegroup consisting of steroids, corticosteroids, and nonsteroidalanti-inflammatory drugs.

Also provided are kits for performing any of the methods providedherein. In some embodiments, kits may include one or more γc-antagonistaccording to any of the embodiments provided herein. In someembodiments, the kit may include instructions. Instructions may be inwritten or pictograph form, or may be on recorded media including audiotape, audio CD, video tape, DVD, CD-ROM, or the like. The kits maycomprise packaging.

Definitions

As used herein, the term “patient” refers to the recipient of atherapeutic treatment and includes all organisms within the kingdomanimalia. In preferred embodiments, the animal is within the family ofmammals, such as humans, bovine, ovine, porcine, feline, buffalo,canine, goat, equine, donkey, deer, and primates. The most preferredanimal is human.

As used herein, the term “treat” or any variation thereof (e.g.,treatment, treating, etc.), refers to any treatment of a patientdiagnosed with a biological condition, such as CD4-, CD8-, andLGL-leukemia, an autoimmune disease, systemic lupus erythematosis,Sjögren's syndrome, Wegener's granulomatosis, Celiac disease,Hashimoto's thyroiditis, a collagen disease, rheumatoid arthritis,inflammatory bowel disease, diabetes mellitus, psoriasis, a degenerativeneuronal disease, multiple sclerosis, uvietis, inflammation of the eye,graft-versus-host disease (GvHD), myasthenia gravis, 1-Human T-cellLymphotropic type I and II (HTLV-I and HTLV-II)-associated diseases,Adult T-cell Leukemia (ATL), HTLV-associated Myelopathy/Tropical SpasticParaparesis (HAM/TSP), uveitis (HU), arthropathy, pneumopathy,dermatitis, exocrinopathy, myositis, influenza, AIDS, HBV, Herpes,asthma, sinusitis, hay fever, bronchitis, chronic obstructive pulmonarydisease (COPD), allergic rhinitis, acute and chronic otitis, lungfibrosis, NK leukemia/lymphoma and NK-T leukemia/lymphoma.

The term treat, as used herein, includes: (i) preventing or delaying thepresentation of symptoms associated with the biological condition ofinterest in an at-risk patient who has yet to display symptomsassociated with the biological condition; (ii) ameliorating the symptomsassociated with the biological condition of interest in a patientdiagnosed with the biological condition; (iii) preventing, delaying, orameliorating the presentation of symptoms associated with complications,conditions, or diseases associated with the biological condition ofinterest in either an at-risk patient or a patient diagnosed with thebiological condition: (iv) slowing, delaying or halting the progressionof the biological condition; and/or (v) preventing, delaying, slowing,halting or ameliorating the cellular events of inflammation.

The term “symptom(s)” as used herein, refers to common signs orindications that a patient is suffering from a specific condition ordisease.

The term “effective amount,” as used herein, refers to the amountnecessary to elicit the desired biological response. In accordance withthe present embodiments, an effective amount of a γc-antagonist is theamount necessary to provide an observable effect in at least onebiological factor for use in treating a biological condition.

“Recombinant DNA technology” or “recombinant” refers to the use oftechniques and processes for producing specific polypeptides frommicrobial (e.g., bacterial, yeast), invertebrate (insect), mammaliancells or organisms (e.g., transgenic animals or plants) that have beentransformed or transfected with cloned or synthetic DNA sequences toenable biosynthesis of heterologous peptides. Native glycosylationpattern will only be achieved with mammalian cell expression system.Prokaryotic expression systems lack the ability to add glycosylation tothe synthesized proteins. Yeast and insect cells provide a uniqueglycosylation pattern that may be different from the native pattern.

A “Nucleotide sequence” refers to a polynucleotide in the form of aseparate fragment or as a component of a larger DNA construct that hasbeen derived from DNA or RNA isolated at least once in substantiallypure form, free of contaminating endogenous materials and in a quantityor concentration enabling identification, manipulation, and recovery ofits component nucleotide sequences by standard molecular biology methods(as outlined in Current Protocols in Molecular Biology).

“Recombinant expression vector” refers to a plasmid comprising atranscriptional unit containing an assembly of (1) a genetic element orelements that have a regulatory role in gene expression includingpromoters and enhances, (2) a structure or coding sequence that encodesthe polypeptide according to the present embodiments, and (3)appropriate transcription and translation initiation sequence and, ifdesired, termination sequences. Structural elements intended for use inyeast and mammalian system preferably include a signal sequence enablingextracellular secretion of translated polypeptides by yeast or mammalianhost cells.

“Recombinant microbial expression system” refers to a substantiallyhomogenous monoculture of suitable hot microorganisms, for example,bacteria such as E. coli, or yeast such as S. cerevisiae, that havestably integrated a recombinant transcriptional unit into chromosomalDNA or carry the recombinant transcriptional unit as a component of aresidual plasmid. Generally, host cells constituting a recombinantmicrobial expression system are the progeny of a single ancestraltransformed cell. Recombinant microbial expression systems will expressheterologous polypeptides upon induction of the regulatory elementslinked to a structural nucleotide sequence to be expressed.

As used herein, the section headings are for organizational purposesonly and are not to be construed as limiting the described subjectmatter in any way. All literature and similar materials cited in thisapplication, including but not limited to, patents, patent applications,articles, books, treatises, and internet web pages are expresslyincorporated by reference in their entirety for any purpose. Whendefinitions of terms in incorporated references appear to differ fromthe definitions provided in the present teachings, the definitionprovided in the present teachings shall control. It will be appreciatedthat there is an implied “about” prior to the temperatures,concentrations, times, etc discussed in the present teachings, such thatslight and insubstantial deviations are within the scope of the presentteachings herein.

Although this invention has been disclosed in the context of certainembodiments and examples, those skilled in the art will understand thatthe present invention extends beyond the specifically disclosedembodiments to other alternative embodiments and/or uses of theinvention and obvious modifications and equivalents thereof. Inaddition, while several variations of the invention have been shown anddescribed in detail, other modifications, which are within the scope ofthis invention, will be readily apparent to those of skill in the artbased upon this disclosure.

It is also contemplated that various combinations or sub-combinations ofthe specific features and aspects of the embodiments may be made andstill fall within the scope of the invention. It should be understoodthat various features and aspects of the disclosed embodiments can becombined with, or substituted for, one another in order to form varyingmodes or embodiments of the disclosed invention. Thus, it is intendedthat the scope of the present invention herein disclosed should not belimited by the particular disclosed embodiments described above.

It should be understood, however, that this detailed description, whileindicating preferred embodiments of the invention, is given by way ofillustration only, since various changes and modifications within thespirit and scope of the invention will become apparent to those skilledin the art.

EXAMPLES

The following Examples are presented for the purposes of illustrationand should not be construed as limitations.

Example 1—Method for Assessing the Inhibitory Activity of γc-AntagonistPeptide

The capacity of any custom derivative peptide prepared according to thepresent embodiments for inhibiting the action of one γc-cytokine familymember is determined using mammalian cellular assays to measure theirproliferative response to the γc-cytokine family member.

For each of the six γc-cytokines, indicator cell lines: CTLL-2, a murineCD8 T cells line available from American Type Culture Collection, andPT-18, a murine mast cell line and its subclone PT-18β, is transfectedwith human IL-2Rβ gene to make the cells responsive to IL-2 and IL-15(Tagaya et al., 1996, EMBO J. 15:4928-39), and is used to quantitativelydetermine the γc-cytokine's growth-promoting activity (See Currentprotocols in Immunology from Wiley and Sons for a methodologicalreference). The indicator cells demonstrate semi-linear dose-dependentresponse when measured by a colorimetric WST-1 assay over a range ofconcentrations (See Clontech PT3946-1 and associated user manual,incorporated herein by reference, for a detailed description of thereagents and methods).

Once the appropriate doses of the cytokine that yield the 50% and 95%maximum response from the indicator cell line is determined, variousconcentrations (ranging from 1 pM to 10 μM) of the purified orsynthesized custom derivative peptide is added to each well containingthe cytokine and indicator cells. The reduction in light absorbance at450 nm is used as an indicator of inhibition of cytokine-stimulatedcellular proliferation. Typically, the cells are stimulated by thecytokines such that the absorbance of the well containing indicator cellline and the cytokine is between 2.0 and 3.0, which is reduced to arange of 0.1 to 0.5 by the addition of inhibitory peptides.

Example 2—BNZ-γ Peptide Specifically Inhibits the Growth-PromotingActivities of IL-9 and IL-15

Using PT-183 cells as described above, the ability of the BNZ-γ peptideto specifically inhibit the growth-promoting activity of selectγc-cytokines was determined (FIG. 3A). IL-3, a non-γc-cytokine thatsupports the growth of PT-183 cells, was used as a negative control.Briefly, PT-185 cells were incubated either with two different dilutionsof BNZ-γ peptide produced by HEK293T cells (1:20 or 1:50 dilution of theoriginal supernatant of HEK293T cells transfected with a BNZ-γexpression construct) or without BNZ-γ peptide in the presence of IL-3,IL-9, IL-15, or IL-4 (1 nM of each cytokine in the culture).

The growth-responses of the cells were determined 2 days after theintroduction of BNZ-γ peptide and the cytokine using the WST-1 assay.The growth-promoting activity of IL-3 (α non γc-cytokine) was notinhibited by BNZ-γ. In contrast, the activity of IL-15 and IL-9 weresignificantly (p<0.01 Student's T test) reduced by the BNZ-γ peptide.Cellular proliferation stimulated by IL-4, another γc-cytokine, was notaffected by the by the addition of BNZ-γ peptide. Results for IL-3,IL-9, IL-15, and IL-4 are shown at FIG. 3A.

In a similar assay, the murine cell line CTTL2 was used. In this assaythe cells were cultured with 0.5 nM of recombinant IL-2 in RPMI 10%fetal Calf Serum. To set up the proliferation assay, cells were washedfrom the cytokines 3 times. Cells were seeded at 1×10(5) cells per wellof a 96-well plate with final concentration of 50 pM of IL-2 or IL-15.Various concentration of BNZ-γ peptide (0.1, 1, and 10 ug/ml) was addedto each well. Cells were cultured for 20 hours and in the last 4 hours,³H-thymidine was added to the plates. Cells were harvested using a platereader. The data is shown in FIG. 3B.

Example 3—Method for Measuring Inhibition γc-Cytokine Activity byAssaying 3H-Thymidine Incorporation of as a Marker of CellularProliferation

Inhibition of γc-cytokine-induced proliferation of an indicator cellpopulation by antagonist custom derivative peptides is measured by the3H-thymidine incorporation assay. Briefly, radiolabeled thymidine (1microCi) is given to 20-50.000 cells undergoing proliferation in thepresence of cytokines. The cell-incorporated radioactivity is measuredby trapping cell-bound radioactivity to a glass-fiber filter using aconventional harvester machines (for example, Filtermate UniversalHarvester from Perkin-Elmer), after which the radioactivity is measuredusing a b-counter (for example, 1450 Trilux microplate scintillationcounter).

Example 4—Method for Measuring Inhibition γc-Cytokine Activity byAssaying Incorporation of a Cell-Tracker Dye as a Marker of CellularProliferation

Indicator cells are incubated in the presence of a selected γc-cytokineor in the presence of a selected γc-cytokine and a selected customderivative peptide. The cell population is then labeled in vitro using acell-tracker dye, for example, CMFDA, C2925 from Invitrogen, and thedecay of cellular green fluorescence at each cellular division ismonitored using a flow-cytometer (for example, Beckton-DickinsonFACScalibur). Typically, in response to γc-cytokine stimulation 7-10different peaks corresponding to the number of divisions that the cellshave undergone will appear on the green fluorescence channel. Incubationof the cells with the selected γc-cytokine and antagonist customderivative peptide reduces the number of peaks to only 1 to 3, dependingon the degree of the inhibition.

Example 5—Inhibition of Intracellular Signaling by BNZ-γ and itsDerivative Antagonists

In addition to stimulating cellular proliferation, binding of theγc-cytokines to their receptors causes a diverse array of intracellularevents. (Rochman et al. 2009 Nat. Rev. Immunol. 9:480-90, Pesu et al.2005 Immunol. Rev. 203:127-142.) Immediately after the cytokine binds toits receptor, a tyrosine kinase called Jak3 (Janus-kinase 3) isrecruited to the receptor at the plasma membrane. This kinasephosphorylates the tyrosine residues of multiple proteins including theγc-subunit, STAT5 (Signal Transducer and Activator of Transcription 5)and subunits of the PI3 (Phosphatidylinositol 3) kinase. Among these,the phosphorylation of STAT5 has been implicated in many studies asbeing linked to the proliferation of cells initiated by the γc-cytokine.(Reviewed in Hennighausen and Robinson, 2008 Genes Dev. 22:711-21.) Inaccordance with these published data, whether or not the BNZ-γ peptideinhibits the tyrosine phosphorylation of STAT5 molecule in PT-18β cellsstimulated by IL-15 was examined (results shown in FIG. 3C).

PT-18β cells were stimulated by IL-15 in the presence or absence ofBNZ-γ peptide. Cytoplasmic proteins were extracted from the cellsaccording to a conventional method as described in Tagaya et al. 1996EMBO J. 15:4928-39. The extracted cytoplasmic proteins were resolvedusing a standard SDS-PAGE (Sodium Dodecyl-Sulfate PolyAcrylamide GelElectrophoresis) and the phorphorylation status was confirmed by ananti-phospho-STAT5 antibody (Cell Signaling Technology, Catalog #9354,Danvers Mass.) using immunoblotting (see FIG. 3C, top panel). To confirmthat each lane represented a similar total protein load, the membranewas then stripped, and re-probed with an anti-STAT5 antibody (CellSignaling Technology, Catalog #9358) (see FIG. 3C, bottom panel).

These results demonstrated that tyrosine phosphorylation of STAT5, amarker of signal transduction, was induced by IL-15 in PT-18β cells, andtyrosine phosphorylation of STAT5 was markedly reduced by the BNZ-γpeptide.

Example 6—Rational Design for BNZ-γ Derivative Antagonistic Peptides

Derivative peptides are prepared based from the core sequenceD/E-F-L-E/Q/N-S/R-X-I/K-X-L/I-X-Q (SEQ ID NO: 2) (where X denotes anyamino acid) by substituting the defined amino acids of the core sequencewith amino acids having identical physico-chemical properties asdesignated in FIG. 2.

Alternatively, custom peptides or their derivative peptides can beprepared based on the sequence alignment of the D-helix regions ofdifferent γc-cytokine family members. For example, as shown in FIG. 5,one or more sequences conserved in γc-cytokine family (SEQ ID NO: 4-SEQID NO: 9) members can be combined to form a peptide such as SEQ ID NO:3.

Example 7—Method of Identifying the Inhibitory Specificity ofAntagonistic Custom Derivative Peptides

The γc-cytokine inhibitory specificity of antagonistic custom derivativepeptides is determined by assaying the ability of a custom derivativepeptide to inhibit the proliferative response of a cytokine-responsivecell line to each of the 6 γc-cytokines. For example, a mouse cell line,CTLL-2, is used to determine if a candidate peptide inhibits thefunction of IL-2 and IL-15. PT-18(β) cells are used to determine if acandidate peptide inhibits the function of IL-4 and IL-9. PT-18 (7α)cells are used to determine if a candidate peptide inhibits the functionof IL-7, and PT-18(21α) cells are used to determine if a candidatepeptide inhibits the function of IL-21. PT-18(β) denotes a subclone ofPT-18 cells that exogenously express human IL-2Rβ by gene transfection(See Tagaya et al. 1996), PT-18(7α) denotes a subclone that expresseshuman IL-7Rα by gene transfection and PT-18(21Rα) cells express humanIL-21Rα.

Another alternative is to use other cell lines that respond to an arrayof cytokines. An example of this cell line in a human NK cell line NK92that is commercially available by ATCC (catalog # CRL-2407). This cellline is an IL-2 dependent cell line that responds to other cytokinesincluding IL-9, IL-7, IL-15, IL-12, IL-18, IL-21 (Gong et al. 1994Leukemia 8: 652-658, Kingemann et al., 1996, Biol Blood MarrowTransplant 2:68; 75, Hodge D L et al., 2002 J. Immunol. 168:9090-8)

Example 8—Preparation of γc-Antagonist Peptides

Custom derivative γc-antagonist peptides are synthesized chemically bymanual and automated processes.

Manual Synthesis:

Classical liquid-phase synthesis is employed, which involves couplingthe carboxyl group or C-terminus of one amino acid to the amino group orN-terminus of another. Alternatively, solid-phase peptide synthesis(SPPS) is utilized.

Automated Synthesis:

Many commercial companies provide automated peptide synthesis for acost. These companies use various commercial peptide synthesizers,including synthesizers provided by Applied Biosystems (ABI). Customderivative γc-antagonist peptides are synthesized by automated peptidesynthesizers.

Example 9—Biological Production of Custom Derivative γc-AntagonistPeptides Using Recombinant Technology

A custom derivative γc-antagonist peptides is synthesized biologicallyas a pro-peptide that consists of an appropriate tagging peptide, asignal peptide, or a peptide derived from a known human protein thatenhances or stabilizes the structure of the BNZ-γ peptide or a peptidecomprising the sequence of SEQ ID NO: 3 or a derivative thereof, andimproves their biological activities. If desired, an appropriateenzyme-cleavage sequence proceeding to the N-terminus of the peptideshall be designed to remove the tag or any part of the peptide from thefinal protein.

A nucleotide sequence encoding the custom derivative peptide with a stopcodon at the 3′ end is inserted into a commercial vector with a tagportion derived from thioredoxin of E. coli and a special peptidesequence that is recognized and digested by an appropriate proteolyticenzyme (for example, enterokinase) intervening between the tag portionand the nucleotide sequence encoding the custom derivative peptide andstop codon. One example of a suitable vector is the pThioHis plasmidavailable from Invitrogen, Calif. Other expression vectors may be used.

Example 10—Conjugation of Custom Peptides and Derivative to CarrierProteins for Immunization Purposes and Generation of Antibody Againstthe Custom Peptides

BNZ-γ and other custom derivative peptides, such as a peptide comprisingthe sequence of SEQ ID NO: 3 or a derivative thereof are used toimmunize animals to obtain polyclonal and monoclonal antibodies.Peptides are conjugated to the N- or the C-terminus of appropriatecarrier proteins (for example, bovine serum albumin, Keyhold LimpetHemocyanin (KLH), etc.) by conventional methods using Glutaraldehyde orm-Maleimidobenzoyl-N-Hydroxysuccinimide Ester. The conjugated peptidesin conjunction with an appropriate adjuvant are then used to immunizeanimals such as rabbits, rodents, or donkeys. The resultant antibodiesare examined for specificity using conventional methods. If theresultant antibodies react with the immunogenic peptide, they are thentested for the ability to inhibit individual γc-cytokine activityaccording to the cellular proliferation assays described in Examples1-3. Due to the composite nature of the derivative peptides it ispossible to generate a single antibody that recognizes two differentcytokines simultaneously, because of the composite nature of thesepeptides.

Example 11—Method for Large Scale Production of Custom Derivativeγc-Antagonist Peptides

Recombinant proteins are produced in large scale by the use of cell-freesystem as described elsewhere. (See Takai et al., 2010 Curr. Pharm.Biotechnol. 11(3):272-8.) Briefly, cDNAs encoding the γc-antagonistpeptide and a tag are subcloned into an appropriate vector (See Takai etal., 2010 Curr. Pharm. Biotechnol. 11(3):272-8), which is subjected toin vitro transcription, followed immediately by an in vitro translationto produce the tagged peptide. The pro-polypeptide is then purifiedusing an immobilized antibody recognizing the tagged epitope, treated bythe proteolytic enzyme and the eluate (which mostly contains the customderivative peptide of interest) is tested for purity using conventional18% Tricine-SDS-PAGE (Invitrogen) and conventional comassie staining.Should the desired purity of the peptide not be met (>98%), the mixtureis subjected to conventional HPLC (high-performance liquidchromatography) for further purification.

Example 12—Use of Custom Derivative γc-Antagonist Peptides to BlockCytokine Function in HAM/TSP

HTLV-1-associated myelopathy (HAM)/tropical spastic paraparesis (TSP) isa chronic progressive myelopathy seen in some people infected with HumanT-Lymphotropic Virus Type I (HTLV-I). Infiltration of lymphocytes in thespinal cord is associated with the immune response to HTLV-I and resultsin the release of certain cytokines. Some of these cytokines may alsodamage nerves.

Patients with HAM/TSP show an elevated state of the immune system thatis similar to that observed in autoimmune diseases (Oh et al. 2008Neurol Clin. 26:781-785). This elevated state is demonstrated by theability of HAM/TSP patient's T-cells to undergo spontaneousproliferation in an ex vivo culture for about a week in the absence ofexogenously added cytokines. The spontaneous proliferation of T-cells inHAM/TSP patients is attributed, at least partly, to autocrine/paracrineloops of IL-2, IL-9, and IL-15. It has been shown that adding blockingantibody against the IL-2 or IL-15 receptors can inhibit spontaneousT-cell proliferation in a HAM/TSP ex vivo culture system.

These observations, along with other data derived from ex vivo studies,have provided the rationale for taking two monoclonal antibodies (ananti-IL-2 receptor alpha or anti-Tac and an anti-IL-15 receptor betachain) into the clinic for treatment of HAM/TSP (Azimi et al. 2001 Proc.Natl. Acad. Sci. 98:14559-64., Azimi et al., 1999 J. Immunol163:4064-72).

Anti-cytokine receptor antagonists according to the embodimentsdescribed herein, would not only be valuable as a therapeuticimmuno-modulatory agent for treatment of HAM/TSP, but modulation ofimmune response in HAM/TSP by anti-cytokine receptor antagonistsaccording to the present embodiments acts proof-of-concept for the useof the anti-cytokine receptor antagonists according to the presentembodiments in the treatment of other auto-immune diseases.

To demonstrate the efficacy of custom derivative γc-antagonist peptidesaccording to the embodiments described herein, we tested the ability ofBNZ-γ peptide to block immune response to HTLV-1 in a spontaneous T-cellproliferation assay using a HAM/TSP ex vivo culture system.Proliferation assays were performed on HAM/TSP patient blood sampleswith and without the addition of BNZ-γ. These assays evaluated theability of BNZ-γ to block the function of cytokines, such as IL-2 andIL-15, present in the ex vivo HAM/TSP patient blood culture and preventspontaneous T-cell proliferation in these samples.

In an ex vivo spontaneous T-cell proliferation assay, PBMC from HAM/TSPpatient was cultured at 1×10(6) cells per well of a 96 well plate inRPMI-10% FCS. Increasing concentrations of BNZ-γ peptide were added toeach well. As a control, an irrelevant peptide was used in similarfashion. The cells were incubated in a 37° C. CO2 incubator for 3, 4,and 6 days. The amount of 1 uCi of ³H-thymidine was added to the cells.After an additional 6 hour incubation, cells were harvested theirproliferation rate was measured. The data for a representative HAM/TSPpatient is shown in FIG. 4A-FIG. 4D. As indicated in FIG. 4A-FIG. 4D,BNZ-γ peptide inhibits the spontaneous proliferation of T-cells inHAM/TSP culture at a concentration of about 1 ug/ml.

Other immunological markers were additionally measured in this assay.The percentage of the viral specific CD8 cells was measured during theex vivo culture using viral protein tetramers. The population ofCD4+CD25+ cells, a marker of T-cell activation, as well as Ki67staining, a marker of T-cell proliferation, was monitored in a flowcytometry assay.

Other forms of the conjugated BNZ-γ peptide derivative or a custompeptide comprising the sequence of SEQ ID NO: 3, and a derivativethereof can be used in a similar future assay. They include albumin,BSA, PEG that can be conjugated to the peptide after chemical synthesis.Other biological forms of custom peptides such as the BNZ-γ peptideconjugate or a custom peptide comprising the sequence of SEQ ID NO: 3,and a derivative thereof may include regions of known protein entities(including but not limited to Fc region of human IgG) that are fused tothe custom peptides.

Example 13—Method of Treating Adult T-Cell Leukemia (ATL) in a HumanPatient by Administration of Custom Derivative γc-Antagonist Peptide

A human patient suffering from Adult T-cell Leukemia is identified. Aneffective dose, as determined by the physician, of custom derivativeγc-antagonist peptide, for example, BNZ-γ, a custom peptide comprisingthe sequence of SEQ ID NO: 3, or a derivative thereof is administered tothe patient for a period of time determined by the physician. Treatmentis determined to be effective if patient enters remission.

Example 14—Method of Treating HAM/TSP in a Human Patient byAdministration of Custom Derivative γc-Antagonist Peptide

A human patient suffering from HAM/TSP is identified. An effective dose,as determined by the physician, of custom derivative γc-antagonistpeptide, for example, BNZ-γ, a custom peptide comprising the sequence ofSEQ ID NO: 3, or a derivative thereof is administered to the patient fora period of time determined by the physician. Treatment is determined tobe effective if patient's symptoms improve or if the progression of thedisease has been stopped or slowed down.

Example 15—Use of Custom Derivative γc-Antagonist Peptides to BlockCytokine Function

A human patient suffering who is in need of reducing the function of atleast IL-15 and IL-21 is identified. An effective dose, as determined bythe physician, of custom derivative γc-antagonist peptide, for example,a composite peptide comprising the sequence of SEQ ID NO: 3 or aderivative thereof is administered to the patient for a period of timedetermined by the physician. Treatment is determined to be effective ifpatient's symptoms improve or if the progression of the disease has beenstopped or slowed down.

Example 16—Method of Treating Celiac Disease in a Human Patient byAdministration of Custom Derivative γc-Antagonist Peptide

A human patient suffering from Celiac disease is identified. Aneffective dose, as determined by the physician, of custom derivativeγc-antagonist peptide, for example, a composite peptide comprising thesequence of SEQ ID NO: 3 or a derivative thereof is administered to thepatient for a period of time determined by the physician. Treatment isdetermined to be effective if patient's symptoms improve or if theprogression of the disease has been stopped or slowed down.

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What is claimed is:
 1. A composite peptide that comprises amino acidsequences of at least two interleukin (IL) protein gamma-c-box D-helixregions, wherein the composite peptide comprises the amino acid sequenceP-K-E-F-L-E-R-F-V-H-L-V-Q-M-F-I-H-Q-S-L-S (SEQ ID NO: 3).
 2. Thecomposite peptide of claim 1, wherein the composite peptide inhibits theactivity of two or more γc-cytokines selected from the group consistingof IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21.
 3. The composite peptide ofclaims 1 and 2, wherein the composite peptide inhibits the activity ofat least IL-15 and IL-21.
 4. The composite peptide of claims 1-3,further comprising a signal peptide.
 5. The composite peptide of claims1-4, wherein the composite peptide is further conjugated to one or moreadditional moieties at the N-terminus, C-terminus or a side residue ofthe composite peptide.
 6. The composite peptide of claims 1-5, whereinthe one or more additional moieties are selected from the groupconsisting of bovine serum albumin (BSA), albumin, Keyhold LimpetHemocyanin (KLH), Fc region of IgG, a biological protein that functionsas scaffold, an antibody against a cell-specific antigen, a receptor, aligand, a metal ion, and Poly Ethylene Glycol (PEG).
 7. The compositepeptide of claims 1-6, wherein the composite peptide consists of anamino acid sequence having at least 90% sequence identity with SEQ IDNO:
 3. 8. The composite peptide of claims 1-6, wherein the compositepeptide consists of an amino acid sequence having at least 95% sequenceidentity with SEQ ID NO:
 3. 9. A pharmaceutical composition comprising:a therapeutically effective amount of a peptide conjugate, or aderivative thereof; and a pharmaceutically acceptable carrier, diluent,excipient or combination thereof; wherein the peptide conjugate or thederivative thereof modulates the activity of two or more γc-cytokinesselected from the group consisting of IL-2, IL-4, IL-7, IL-9, IL-15, andIL-21; wherein the peptide conjugate comprises the amino acid sequenceP-K-E-F-L-E-R-F-V-H-L-V-Q-M-F-I-H-Q-S-L-S (SEQ ID NO: 3).
 10. Thepharmaceutical composition of claim 9, wherein the peptide conjugate orthe derivative thereof inhibits the activity of two or more γc-cytokinesselected from the group consisting of IL-2, IL-4, IL-7, IL-9, IL-15, andIL-21.
 11. The pharmaceutical composition of claims 9 and 10, whereinthe peptide conjugate or the derivative thereof further comprises anadditional conjugate at the N-termini, C-termini or a side residuesthereof.
 12. The pharmaceutical composition of claims 9-11, wherein thepeptide conjugate or the derivative thereof further comprises a signalpeptide.
 13. The pharmaceutical composition of claims 9-12, furthercomprising a protein that stabilizes the structure of the peptideconjugate or the derivative thereof and improves its biologicalactivity, wherein the protein is selected from the group consisting ofbovine serum albumin (BSA), albumin, Fec region of immunoglobulin G(IgG), biological proteins that function as scaffold, Poly EthyleneGlycol (PEG), and derivatives thereof.
 14. The pharmaceuticalcomposition of claims 9-13, wherein the derivative thereof comprises apeptide sequence sharing at least 90% identity with the amino acidsequence of SEQ ID NO:
 3. 15. The pharmaceutical composition of claims9-13, wherein the derivative thereof comprises a peptide sequencesharing at least 95% identity with the amino acid sequence of SEQ ID NO:3.
 16. A method for blocking signaling by two or more γc-cytokine familymembers, comprising contacting a cell with the composite peptide ofclaims 1-8 and/or the pharmaceutical composition of claims 9-15.
 17. Amethod of inhibiting γc-cytokine binding to a γc-subunit comprisingcontacting a γc-subunit of a cell with the composite peptide of claims1-8 and/or the pharmaceutical composition of claims 9-15.
 18. A methodof treating a γc-cytokine-mediated disease, the method comprisingadministering the composite peptide of claims 1-8 and/or thepharmaceutical composition of claims 9-15 to a subject in need thereof,wherein the γc-cytokine-mediated disease is selected from the groupconsisting of CD4-leukemia, CD8-leukemia, LGL-leukemia, systemic lupuserythematosis, Sjögren's syndrome, Wegener's granulomatosis, Celiacdisease, Hashimoto's thyroiditis, rheumatoid arthritis, diabetesmellitus, psoriasis, multiple sclerosis, uvietis, inflammation of theeye, and graft-versus-host disease (GvHD).
 19. A method of treating anHTLV-1-associated myelopathy (HAM)/tropical spastic paraparesis (TSP)associated disease, the method comprising administering the compositepeptide of claims 1-8 and/or the pharmaceutical composition of claims9-15 to a subject in need thereof, wherein the HAM/TSP associateddisease is selected from the group consisting of Adult T-cell Leukemia(ATL), HTLV-associated Myelopathy/Tropical Spastic Paraparesis(HAM/TSP), and other non-neoplastic inflammatory diseases associatedwith HTLV such as uveitis (HU), arthropathy, pneumopathy, dermatitis,exocrinopathy, and myositis.
 20. A method of treating an inflammatoryrespiratory disease, the method comprising administering the compositepeptide of claims 1-8 and/or the pharmaceutical composition of claims9-15 to a subject in need thereof, wherein the inflammatory respiratorydisease is selected from the group consisting of asthma, sinusitis, hayfever, bronchitis, chronic obstructive pulmonary disease (COPD),allergic rhinitis, acute and chronic otitis, and lung fibrosis.
 21. Amethod of treating a cosmetic condition, the method comprisingadministering the composite peptide of claims 1-8 and/or thepharmaceutical composition of claims 9-15 to a subject in need thereof,wherein the cosmetic disease is selected from the group consisting ofacne, hair loss, sunburn, nail maintenance, and appearance of aging. 22.A pharmaceutical composition for use in treating a condition in apatient, the pharmaceutical composition comprising: a therapeuticallyeffective amount of a peptide conjugate, or a derivative thereof; and apharmaceutically acceptable carrier, diluent, excipient or combinationthereof; wherein the peptide conjugate or the derivative thereofmodulates the activity of two or more γc-cytokines selected from thegroup consisting of IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21; whereinthe peptide conjugate comprises the amino acid sequenceP-K-E-F-L-E-R-F-V-H-L-V-Q-M-F-I-H-Q-S-L-S (SEQ ID NO: 3).
 23. Thepharmaceutical composition of claim 22, wherein the derivative thereofcomprises a peptide sequence sharing at least 90% identity with theamino acid sequence of SEQ ID NO:
 3. 24. The pharmaceutical compositionof claim 22, wherein the derivative thereof comprises a peptide sequencesharing at least 95% identity with the amino acid sequence of SEQ ID NO:3.
 25. The pharmaceutical composition of claims 22-24 for use intreating a γc-cytokine-mediated disease, wherein theγc-cytokine-mediated disease is selected from the group consisting ofCD4-leukemia, CD8-leukemia, LGL-leukemia, systemic lupus erythematosis,Sjögren's syndrome, Wegener's granulomatosis, Celiac disease,Hashimoto's thyroiditis, rheumatoid arthritis, diabetes mellitus,psoriasis, multiple sclerosis, uvietis, inflammation of the eye, andgraft-versus-host disease (GvHD).
 26. The pharmaceutical composition ofclaims 22-24 for use in treating an HTLV-1-associated myelopathy(HAM)/tropical spastic paraparesis (TSP) associated disease, wherein theHAM/TSP associated disease is selected from the group consisting ofAdult T-cell Leukemia (ATL), HTLV-associated Mycelopathy/TropicalSpastic Paraparesis (HAM/TSP), and other non-neoplastic inflammatorydiseases associated with HTLV such as uveitis (HU), arthropathy,pneumopathy, dermatitis, exocrinopathy, and myositis.
 27. Thepharmaceutical composition of claims 22-24 for use in treating aninflammatory respiratory disease, wherein the inflammatory respiratorydisease is selected from the group consisting of asthma, sinusitis, hayfever, bronchitis, chronic obstructive pulmonary disease (COPD),allergic rhinitis, acute and chronic otitis, and lung fibrosis.
 28. Thepharmaceutical composition of claims 22-24 for use in treating acosmetic condition, wherein the cosmetic disease is selected from thegroup consisting of acne, hair loss, sunburn, nail maintenance, andappearance of aging.
 29. A kit for treating a condition in a patient,wherein the condition is a ye cytokine-mediated disease, anHTLV-1-associated myelopathy (HAM)/tropical spastic paraparesis (TSP)associated disease, an inflammatory respiratory disease, a cosmeticcondition, or a combination thereof, the kit comprising a pharmaceuticalcomposition of claims 9-15 and/or claims 22-28, wherein thepharmaceutical composition comprises: a therapeutically effective amountof a peptide conjugate, or a derivative thereof; and a pharmaceuticallyacceptable carrier, diluent, excipient or combination thereof; whereinthe peptide conjugate or the derivative thereof modulates the activityof two or more γc-cytokines selected from the group consisting of IL 2,IL 4, IL 7, IL 9, IL 15, and IL 21; wherein the peptide conjugatecomprises the amino acid sequenceP-K-E-F-L-E-R-F-V-H-L-V-Q-M-F-I-H-Q-S-L-S (SEQ ID NO: 3).
 30. The kit ofclaim 29, wherein the derivative thereof comprises a peptide sequencesharing at least 90% identity with the amino acid sequence of SEQ ID NO:3.
 31. The kit of claim 29, wherein the derivative thereof comprises apeptide sequence sharing at least 95% identity with the amino acidsequence of SEQ ID NO:
 3. 32. The kit of claims 29-31, wherein thecondition is one or more of CD4 leukemia, CD8 leukemia, LGL leukemia,systemic lupus erythematosis, Sjögren's Sjoegren's syndrome, Wegener'sgranulomatosis, Celiac disease, Hashimoto's thyroiditis, rheumatoidarthritis, diabetes mellitus, psoriasis, multiple sclerosis, uvietis,inflammation of the eye, graft-versus-host disease (GvHD), Adult T-cellLeukemia (ATL), HTLV-associated Myelopathy/Tropical Spastic Paraparesis(HAM/TSP), and other non-neoplastic inflammatory diseases associatedwith HTLV such as uveitis (HU), arthropathy, pneumopathy, dermatitis,exocrinopathy, myositis, asthma, sinusitis, hay fever, bronchitis,chronic obstructive pulmonary disease (COPD), allergic rhinitis, acuteand chronic otitis, and lung fibrosis, acne, hair loss, sunburn, nailmaintenance, or appearance of aging.